Introduction

In vitro cytotoxicity assays provide preliminary
information about the activity of an antineoplastic agent against a
particular solid tumor, but these techniques are limited by the
difficulties in obtaining and culturing human cells in explant. It may
take weeks to determine the success or failure of a specific
chemotherapeutic regime in a patient, if the response to antineoplastic
activity is based on change in size of a soft tissue lesion. Therefore,
some patients have ineffective drugs administered needlessly until
clear progression is observed in a computed tomography scan or proton
MRI3
or clinical
symptoms worsen. Apoptosis and related cellular changes, however, can
be observed in vitro within hours of exposure to
antineoplastic agents. An in vivo assay of chemotherapeutic
efficacy based on such rapid changes would significantly contribute to
patient management by providing real-time drug efficacy information so
that therapy could be optimized and ineffective therapy discontinued.
Hence, various recent studies have focused on this problem using
changes in F-18 fluorodeoxyglucose uptake [as measured with positron
emission tomography imaging (1)]
and changes in cell
metabolism [as measured with P-31 MR spectroscopy
(2, 3, 4)]
in an effort to obtain noninvasive, real-time
information on therapeutic effect. However, being able to accomplish
this goal using higher resolution magnetic resonance imaging would have
enormous clinical benefit. The novel application of MRI that we
describe uses sodium nuclei and weights sodium MR images toward
populations of sodium nuclei that are physiologically relevant to
detecting tumors and monitoring their treatment. In addition to
detecting rapid cellular events, this sodium MRI approach has the
additional advantage of ready coregistration with high resolution
proton MRI, allowing for comparison with tissue structure, angiography,
and imageable gene markers.

Because of the biological importance of sodium, its relative abundance,
and its sensitivity, sodium NMR is a particularly useful tool for the
study of pathophysiological processes. The large transmembrane ionic
gradient causes the much lower IC concentration
([Na]i) to be highly dependent on active
processes (Na+/K+-ATPase
pump) and thus responsive to metabolic suppression. With special
relevance to the study of neoplasms, [Na]i is
correlated with the proliferation rate of nonneoplastic and malignant
cell populations (5)
, presumably because of the role of
Na+ influx in initiating movement through the
cell cycle. Na+ flux is mediated by transmembrane
exchangers for both Ca2+ and
H+(6, 7)
. Thus, not only is there
probable elevation of [Na]i because of the
toxic effects of chemotherapy, but the altered values of baseline[
Na]i, if detectable, could also have
diagnostic significance.

Measurement of sodium content clinically has typically been done using
SQ NMR techniques (8, 9)
. A significant disadvantage of SQ
NMR is the relatively larger abundance of EC versus IC[
Na]. SQ NMR requires paramagnetic shift reagents to discern
Nai. Because of their relative membrane
impermeability, they selectively shift the MR spectrum of the EC
sodium. However, shift reagents would have severe disadvantages,
including toxicity, in clinical use. An alternative MR approach to
measure IC sodium content is based on the interaction of sodium
polyanions and their resultant effects on nuclear spin transitions.
Spin 3/2 nuclei (such as Na and K) have a nonvanishing
quadrupole moment, allowing interaction with electrostatic field
gradients (10)
. In certain complex environments, such as
those occurring in IC space, MQ spin transitions occur that can be
detected by specific pulse sequences called MQ filters
(11, 12, 13, 14)
. The EC sodium diffuses relatively freely, with a
small but presumed constant component electrostatically bound to plasma
proteins and the surface of cell membranes. Thus, the presence of an MQ
signal can be used to identify populations of sodium nuclei by their
molecular environment and can be used to detect[
Na]i changes (15, 16, 17, 18, 19, 20, 21, 22)
.

In this study, we capture this utility of MQ sodium MRI, the ability to
identify sodium by its microenvironment, but use instead a SQ pulse
sequence. The higher signal-to-noise of SQ sodium MRI is a distinct
clinical advantage. The sodium subpopulation weighting is accomplished
by applying an IR pulse sequence to null the signal from sodium nuclei
with long longitudinal relaxation times
(T1), as found in free solution or
plasma. The image signal is then weighted toward bound sodium, which is
primarily IC. The IR technique is commonly used in proton imaging to
distinguish fat and water. This study was designed to test the adequacy
of such a weighting scheme and whether images so derived would have the
hypothesized diagnostic utility.

For [Na]i, an in situ calibration
technique was used whereby cells were permeabilized to
Na+ with monensin (20 μm)
and gramicidin (40 μm);[
Na]o was varied in EC solutions while
adjusting [K]o, [Cl]o,
and isothionate as described (27, 28)
. For[
Ca]i measurements, one row of wells was
exposed to ionomycin (10 μm) and one to
ionomycin plus EGTA (20 mm) to derive
Rmax and
Rmin,
(Kd was assumed to be 371
nm). The average control concentration for the
five experiments was 126 nm. We displayed the
percentage of change in control concentration calculated from the ratio
measurements (26)
. For ion measurements, plates were run
on a Fluoroskan II fluorescent plate reader (Titertek). Ratios were
derived by dividing fluorescent intensity at 345 nm excitation by that
at 390 nm, both with a 508-nm emission filter. For all plates, dye-free
wells were examined for both control and drug-treated cells and served
as background values. For [Ca]i experiments,
exposure to 6 mm MnCl2,
which quenches dye, was an added control (26)
. Averaged
background values were subtracted from each well in the protocol
grouping prior to deriving the ratio. Thapsigargin was added as a
positive control for [Ca]i elevation and
generated rapid elevations in excess of 1 μm.

Two reference phantoms of 200 mmol/l NaCl with either 30% (P1) or 40%
Ficoll (P2) were glued to a plastic animal platform. The anesthetized
mouse was aligned in a deep grove with the tumor positioned through a
small elliptical opening in the plastic holder, thus maintaining the
same relative position to the phantoms from experiment to experiment.
The 40% phantom is the brightest phantom on the IR image, and the 30%
phantom is the brightest on the SQ image. IR images and SQ images were
normalized to the brightest phantom within each slice.

Analysis of tumor intensity change was measured at the brightest
region using line profiles or region-of-interest resident software.
This was generally at the center for uniformly bright images or on the
largest portion of the annulus. To confirm tumor and kidney positions,
paired proton and sodium quadrature birdcage coils, identical in
dimension, were used.

Administration of Chemotherapy.

Drug was dissolved in 100–150 μl of vehicle and slowly injected into
a femoral vein under microscopic observation and while the mouse was
anesthetized with ketamine and xylazine. We injected 10 mg/ml Taxotere
(i.e., ∼1.0 mg) or 2 mg/ml VP-16 (∼0.3 mg). The reported
toxic mouse dose for Taxotere is 4.5 mg. The mouse doses equivalent to
human doses were calculated from standard equations that convert weight
to surface area for small mammals. Because of problems with viscosity
for the mouse i.v. injections, the VP-16 dose was about one-third the
calculated equivalent human dose. All animals were used in accordance
with Institutional Animal Care and Use Committee and Columbia
University rules for humane treatment of animals.

Tumor Postmortem.

Formalin-fixed, paraffin-embedded sections of the explanted neoplasms
were cut at 4 μm and stained with H&E or deparaffined and
fluorescently end-labeled (100-μl aliquots of APO-BrdU kit reagents
applied directly to slides in a moist environment). The number of
mitotic figures was enumerated in up to 20 HPFs (×400) in the rim of
viable cancer cells for each specimen. Care was taken not to include
central areas with cellular debris or foci with back-to-back apoptotic
cells. In some specimens, <20 HPFs were available for evaluation. The
mitotic index represents the average number of mitotic figures per HPF
(29, 30)

Results

Sodium Inversion Recovery Can Null Signal from Selected
T1 Range.

To produce the weighted sodium image, an IR pulse sequence is used to
suppress the signal from sodium nuclei with long
T1 relaxation times (corresponding to
free sodium). This technique selects populations of sodium nuclei based
on longitudinal relaxation time (T1),
as opposed to the transverse relaxation time
(T2) used in the standard MQ approach.
In general, to suppress the contribution of sodium nuclei with a range
of T1 values about some
T1ex, one sets the
inversion time (the time between the 180° and 90° pulses in the IR
pulse sequence) to (ln
2)(T1ex). This is a
good approximation for long repetition times. Because of the higher
concentration of total sodium and lower proportion of bound sodium in
the EC space, an IC weighted sodium signal and image are produced.

The ability to weight for different populations of sodium nuclei is
easily demonstrated using a cell-free system, e.g.,
phantoms. The phantoms were tubes of NaCl solution. Some contained Agar
or Ficoll to simulate the increased viscosity and electrostatic binding
of sodium, which occurs to some extent in the EC space but more
extensively in the IC space. With appropriate choice of inversion time,
depending on the concentration of Agar or Ficoll, the signal from a
particular test phantom can be totally suppressed. We imaged two
phantoms (with and without 4% Agar) at nine different inversion times,
and in the absence of an inversion pulse, and plotted the intensity for
each image as a separate point. Complete suppression was obtained at
inversion times of 19 and 31 ms, respectively (Fig. 1)<$REFLINK>
. The straight-line is a fit of the
theoretical relationship between image intensity,
T1 and inversion time (assuming
T1 values of 27 and 43 ms). This
exercise demonstrates how one can totally suppress the signal from a
particular homogeneous population of sodium nuclei based on relaxation
times.

Effects of inversion time on phantoms. Average
image intensity for two phantoms (1 m NaCl, ○; 1
m NaCl plus 4% agarose, •) were examined during SQ
acquisition and for nine different IR times (horizontal
axis; Tinv). SQ is equivalent to
zero inversion time. The lines are the theoretical relationships
assuming T1s of 27.4 and 43.3 ms (inversion
times of 19 and 31 ms). Intensity of IR signal varies, such as
ABS (1 −
2exp(−Tinv/T1)) .
Inset, a cystic tumor (fluid measured by needle
aspiration) imaged with a 25-ms inversion pulse sequence (right
inset) and normal SQ sequence (left inset). The
cystic tumor was completely eliminated by inversion pulse. The mouse
body contour is delineated in red.

Application of Sodium IR to Mouse Tumors.

To determine the best pulse sequence for suppressing EC sodium,
different inversion times were tested on a human tumor xenograft mouse
model of prostate cancer. We chose 25 ms as the optimum inversion time
for several reasons. For this inversion time, the signal was totally
nulled for a cystic tumor (e.g., a tumor sac that was
predominantly fluid; Fig. 1<$REFLINK>
, insets). Furthermore, the mouse
kidneys, which appeared very bright on the SQ MR image with no
inversion pulse, were also totally suppressed on the IR image (Fig. 2<$REFLINK>
, compare C and
D). This inversion pulse, therefore, was quite effective at
nulling EC bulk fluid.

Effects of IR pulses on tumor images. A
24-slice three-dimensional gradient-echo SQ image and a 24-slice IR
image were acquired in 15 and 45 min, respectively. One slice each from
a DU145 tumor (A and B) and PC3 tumors
(C and D) show noninverted SQ
(left panels, A and C), and IR
acquisitions (right panels, B and D).
Tumors (straight arrows) are bright in all image sets
but are dominant in the IR images. Note that kidneys (C, curved
arrows) are bright in the SQ image but completely suppressed in
IR image (D). The tumor is directly below the mouse
body, which is delineated in red or
yellow, and confirmed by coregistered proton image (not
shown). The phantoms (P1 and P2; see“
Materials and Methods”) have different
T1 values and respond differently to the
inversion sequence. Acquisition parameters are described in“
Materials and Methods.”

This same pulse sequence (using a 25-ms inversion pulse), which
completely suppressed EC fluid signal, was also ideal for
enhancing the visibility of solid tumors, which appeared relatively
brighter in comparison with the mouse body. This is presumably because
of the higher IC sodium in tumor versus normal tissue (see“
Discussion”) and the ability of this pulse sequence to enhance the
contribution of IC sodium. Compare the IR sodium images (Fig. 2, B and D)<$REFLINK>
with traditional (no inversion pulse) SQ
sodium images (Fig. 2, A and C)<$REFLINK>
for solid tumors
formed from two human cell lines (DU145, Fig. 2, A and B<$REFLINK>
; PC3, Fig. 2, C and D<$REFLINK>
). Thus, 25 ms
is the optimal inversion time for both suppressing EC sodium nuclei as
well as relatively enhancing the image of solid tumors at the magnetic
field strength (4.23 Tesla) used in our experiments. Note that the
tumor is also bright on the traditional SQ image (see“
Discussion”).

Intracellular Ion Elevation during Drug-induced Apoptosis in
Culture.

The dissipation of the transmembrane sodium gradient during cell
necrosis has been well studied. Less well studied are changes in ionic
gradients during apoptosis. To examine the relationship between sodium
elevation and the extent of apoptotic cell progression in a controlled
setting, cultured PC3 (and in some cases DU 145) cells were exposed to
the two drugs used in the in vivo experiments,
i.e., Taxotere (which disrupts microtubule assembly), and
VP-16 (a topoisomerase inhibitor). Apoptosis was assessed with flow
cytometry using both fluorescent end-labeling of DNA fragments and
Annexin V binding (Fig. 3, A and B)<$REFLINK>
. [Na]i elevation was examined
using the fluorescent ratiometric dye, SBFI/AM (Fig. 3<$REFLINK>C).
Because Ca2+ is coupled to
Na+ by various membrane exchangers and is linked
to cell death and apoptosis, free concentration
([Ca]i) was also measured using a different
ratiometric dye, Fura II/AM (Fig. 3<$REFLINK>D). Because[
Ca]i elevation would be expected to accompany[
Na]i elevation, this additional measurement
served as an independent confirmation of ionic response.

Chemotherapy, cellular apoptosis, and ion
activity. Representative data from flow cytometry (A and
B) and fluorescent measurements in adherent cells
(C and D) are shown. A,
response at 24 h shows histogram of end-labeling intensity
(24)
representing apoptotic nuclei for the most responsive
cell/drug combination (Du145/VP-16). ×100 increase. B,
the time course of Annexin V fluorescent labeling of propidium iodide
excluding cells for VP-16 (10 μg/ml; ▴) and Taxotere (10
nm; •) is plotted. One-dimensional annexin histograms
(FITC intensity) for live cells are shown for control (bottom
inset) and after 24 h VP-16 (top inset).
C, [Na]i elevation in PC3 cells adherent
to 96-well plates is measured with a fluorescent ratiometric dye,
indicating an elevation of 10–20 mm (from the in
situ calibration technique), which starts as early as 2–6 h
(28)
. D, [Ca]i elevations as
a percentage of control also occurs early and persists. Using standard
techniques with Ca2+ ionophores and buffers, the absolute
value of mean control was estimated at 126 μm, with
average elevations of 150 μm. Each data point in
C and D represents mean of five wells;
bars, SE.

At the concentrations selected (10 nm Taxotere and 10μ
g/ml VP-16), end-labeling fluorescence was not seen at 8 h and
was not fully developed until 24–48 h in PC3 cells. The only rapid
response for end-labeling fluorescence (within 24 h) was seen with
the combination of VP-16 and DU145 cells (Fig. 3<$REFLINK>A). In PC3
cells, the earliest Annexin V response was 5 h (VP-16), but
it required an additional 24 h (VP-16) or 48 h (Taxotere) to
fully develop at the drug concentrations used (Fig. 3<$REFLINK>B). The
ionic elevations (approximated at 10–20 mm for[
Na]i and 150 μm for[
Ca]i) were already significant as early as
2–6 h and peaked at 6–24 h (Fig. 3, C and D)<$REFLINK>
.
Thus, drug-induced ionic elevation preceded the development of
apoptotic markers, suggesting that the ionic responses are not a
trivial manifestation of the loss of membrane integrity associate with
cell death. The rapid ionic responses further indicated that we should
set the earliest in vivo measurement time to no later than
24 h and motivated future experiments to identify the early
apoptotic mechanisms involved.

Increased Intensity of IR Sodium Image during Drug Effects
in Vivo.

The effect of chemotherapy on the IR sodium image intensity of
in vivo tumors was consistent with the culture results,
i.e., IR sodium image intensity increased after in
vivo drug administration. This is suggestive of increased[
Na]i. Control and posttherapy images were
acquired in 16 mice. Tumors in two additional mice had images
reacquired after control saline injections. Taken as a whole, the
tumors exhibited increased intensity (36% ± 4%) after chemotherapy
(P < 0.001; n = 16), a response
significantly different (P < 0.002) than that after
saline injections (−5% ± 4%; n = 4; not significant
versus control at either 24 or 48 h). Taken separately,
the change for each drug on PC3 cells was significant; Taxotere induced
a 32% increase (P < 0.001; n = 5;
Fig. 4<$REFLINK>
, top left) and VP-16
induced a 38% change (P < 0.0005; n =
7; Fig. 4<$REFLINK>
, bottom left). The responses grouped by drug were
not significantly different from each other (P >
0.15). The average signal at 48 h was still elevated compared with
control (28%, P < 0.05; n = 11) for
the PC3 cell tumors. Four experiments using a second human prostate
cancer cell line (DU145; two each with Taxotere and VP-16) also showed
significant elevation at 24 h taken as a group (34%;
P < 0.05; n = 4). An independent
measure of drug effect, reduction in mitotic index, is plotted in the
right panel and is shown to correlate with image enhancement
(see below).

IR image response to drug. After acquisition of
control images, antineoplastic agents were administered. Reimaging
occurred at indicated times. The graph shows increased image intensity
at 24 h versus control for both Taxotere
(top left panel) and VP-16 (bottom left
panel). Graphs show both PC3 tumors (•) and DU145 tumors
(○). Explanted tumors were then removed, fixed (10% formalin), and
stained (H&E). The number of mitotic figures was enumerated in a
maximum of 20 HPFs (×400) for each specimen. Quantitative analysis was
only performed in the most peripheral zone of the neoplasms
representing the viable tumor cells with scattered single cell
apoptosis and excluding nonviable areas or foci with back-to-back
apoptosis. Data are expressed as mitotic index representing the average
number of mitotic figures per HPF. Means for untreated (○) and
treated tumors (•) are plotted (right panel)
versus sodium MRI image change (vertical axes,
left panels); bars, SE. Among treated tumors,
there is a significant negative correlation between mitotic figures and
sodium IR image intensity (P < 0.02), which is
indicated by the regression line. (For acquisition parameters, see“
Materials and Methods.”)

Independent Assay of Drug Effect Using Histological Measure of
Proliferation.

In an effort to determine the origins of the IR image and enhanced
intensity and to verify that the drugs attained therapeutic levels, 10
of the imaged tumors were explanted after final imaging sessions and
examined. On the basis of morphology, the neoplastic tissue was divided
into three concentric zones, which included a central zone with
cellular debris and absence of cell nuclei, surrounded by a rim of
back-to-back apoptotic cells. An outermost peripheral zone was viable
tumor tissue. The border between the viable and nonviable tissue could
be clearly seen in HPFs, where a rim of densely packed apoptotic cells
was clearly visible abutting the surviving rim of viable cells.

In HPFs of the surviving rims of these tumors (Fig. 5)<$REFLINK>
, one can see that the untreated tumor
(Fig. 5<$REFLINK>A) has more mitotic figures than the treated tumor
(Fig. 5<$REFLINK>B). Because a reduction in proliferation,
attributable to cell cycle arrest, is a useful indicator of
antineoplastic efficacy (29, 30)
, the incidence of mitotic
figures was measured for up to 20 HPFs in the surviving rim of each
tumor. Mitotic figures were reduced significantly (P <
0.0001) in the treated (n = 7) tumors [2.24 ±
0.163 (SE) per field; m = 127 fields]
versus untreated (n = 3) tumors (8.57 ± 0.84 per field; m = 47). Importantly, there was,
among all treated tumors, a significant inverse correlation
(P < 0.02) between the reduction in mitotic figures
and the relative enhancement in the IR sodium image (Fig. 4<$REFLINK>
,
right panel).

Change in mitotic index in HPFs. HPFs of the
viable rim of untreated (A) and treated
(B) tumors, stained for H&E, are shown. The
saline-treated tumor (A) shows a greater number of
mitotic figures (arrows) than the neoplasms subjected to
chemotherapy (B). Single apoptotic cells
(circle in B) are easily found in the
treated specimen. ×400.

Correlation of Image Configuration with Histology and
Immunofluorescence.

Because the tumor was explanted in this study after the final imaging
session, which was not always at the point of peak image enhancement,
we are limited in our postmortem analysis. Nevertheless, we can draw
tentative conclusions about the origin of the bright entities on the IR
images.

Pre- and postdrug IR images are shown along with the control SQ image
for a tumor with significant central necrosis. Tumors with significant
central necrosis had a characteristic dark-center/bright-annulus
presentation on the inversion image (Fig. 6, A and B)<$REFLINK>
. These
tumors were nevertheless uniformly bright on the SQ image (no
inversion; Fig. 6<$REFLINK>C). Note that the effect of drug exposure
(enhanced image intensity) is clearly visible comparing the two IR
images but is confined to the enhanced intensity of the annulus. The
dark center on the IR image was unchanged after drug administration.
The histology of the tumor (Fig. 6<$REFLINK>D) shows a significant
central area with nonviable cells and cellular debris and a narrow,
peripheral rim of viable cells (between is a region of back-to-back
apoptosis).

Morphological differentiation based on extent of
nonviable center. A–C, sodium MR images from a
tumor Q with a necrotic center. The noninverted SQ image
(C) is uniformly bright, whereas the IR images
(A and B) have dark centers. IR image
intensity of the annulus increases after drug injection (compare
control A with B). The mouse body is
delineated in red. The carcinoma (also tumor Q) stained
for H&E (lower right panel), showing a narrow rim of
viable tissue (V) and a large nonviable center
(N). The slim interface (arrows) between
viable (V) and necrotic (N) zones shows
apoptotic cells (arrow in inset).
Bar, 1 mm.

In tumors with small or absent nonviable centers, the IR images were
uniformly bright (e.g., tumor M; Fig. 7, A–C<$REFLINK>
). A broad band of
densely apoptotic cells is seen below the surviving rim, and the
nonviable center is comparatively smaller than in Q (Fig. 7<$REFLINK>F, compare with Fig. 6<$REFLINK>D). The border between the
surviving rim, and densely apoptotic cells are shown at high power in
the inset (Fig. 7<$REFLINK>F). Furthermore, on the images there is a
clear increase in the diameter of the area of brightness on the IR
image, which occurred between 24 and 48 h after treatment. We
examined this region of recent IR brightening under higher power.
Evidence of a well-defined rim of apoptotic cells just under the
surviving rim was visible in adjacent tissue sections stained either
with H&E (Fig. 7<$REFLINK>D) or immunofluorescent end-labeling (Fig. 7<$REFLINK>E). We examined the fluorescent staining; the cells just
under the surviving rim were brightest, and the cells closer to the
small nonviable center were less bright. Presumably, the most brightly
stained cells most recently underwent apoptosis, and the less bright
cells had more time to lose their fragmented DNA. Thus, cells with no
nuclear staining on H&E micrographs were present only in the small
center of tumor M (∼300 μm). In contrast, tumor Q showed a
large center of cells without nuclei. On the basis of the image
broadening and immunofluorescence, a plausible interpretation is that a
portion of the inner surviving rim was converted to a zone of
back-to-back apoptotic cells between 24 and 48 h. Thereafter, but
not yet at the time of tumor removal, cellular debris would have
accumulated in the center, as seen with Q.

Further evidence from images and histology after
drug administration. Carcinoma (tumor M) shows a uniformly bright IR
image for control, 24 and 48 h (A–C). The outer
margin of the IR image is seen to expand by ∼100 μm after the 24-h
image but prior to the 48-h image. The mouse body is delineated in
red. The tumor is visualized at the
bottom of the body image, between phantoms. Histology is
shown from tumor (M), indicating a small central area of
nonviable tissue. In H&E staining (D), the surviving rim
of viable cells (V) appears peripheral to a wide region
of back-to-back apoptosis (A). The fluorescently
end-labeled companion slide (E) indicates a
well-demarcated bright region just below the surviving rim, presumably
representing the border with the most recently apoptotic cells
(illustrated at high power in the inset). A low power
field (F) indicates a thick rim of viable tissue
(V) and a small nonviable center (N). The
broad interface (arrows) between viable
(V) and nonviable (N) zones contains
back-to-back apoptotic cells (arrows in
inset). Bar, 1 mm. The
right-hand phantom measures 8 mm in diameter.

Discussion

These results are consistent with reports that ratios of IC
Na+versus K+
are higher in both benign and malignant tumors than in their normal
cellular counterparts (31, 32)
. In vitro tissue
studies further showed that [Na]i is elevated
by as much as a factor of two in neoplastic compared with normal
tissue, malignant compared with benign tumor, and poorly differentiated
compared with well-differentiated tumor (33, 34)
. Because
ionic alterations are important events in malignant transformation,
apoptosis, necrosis, and progression through the cell cycle, it is not
surprising that successful antineoplastic agents affect IC ions
(35, 36)
. Consistent with our results (Figs. 3<$REFLINK>
and 4)<$REFLINK>
,
various antineoplastics (e.g., colchicine, lonidamine, and
VP-16) have been reported to elevate [Na]i
and/or [Ca]i(37, 38, 39, 40)
.

For Taxotere, the in vivo injection doses we used for mice
were comparable with human clinical doses. Furthermore, human plasma
levels of Taxotere after a single loading dose (41)
remain
above the levels (10 nmol/l) used in our tissue culture studies for∼
24 h. Thus, we expect that our tissue culture results (elevation of
both [Na]i and [Ca]i
and significant apoptosis) would apply during clinical in
vivo tumor studies. The VP-16 tissue culture levels we used were
comparable with average human plasma levels during the first 4–6 h
after a standard dose (100 mg/m2; Ref.
42
).

On the basis of our in vivo and tissue culture studies and
in vitro literature reports, the most straightforward
explanation for the increased image intensity after chemotherapy is
increased [Na]i. We have, however, considered
several alternate explanations, which although less likely, still must
be investigated: (a) decreased IC
T1 (or change in EC
T1); (b) increased EC
binding sites; and (c) increased EC[
Na]o. Explanation c seems unlikely,
because even in excitable tissue, where there is a high density of
sodium channels and
Na+/K+-ATPase pump sites,
there is no evidence of bulk changes in [Na]o
beyond the unstirred layers around channel pores. For explanation
b, one may speculate whether drug-induced processes could
alter production of metalloproteinases, affecting the density of EC
sodium binding sites. During hypoxia and
Na+/K+-ATPase pump blockade
in the heart, multiple quantum sodium MRI studies showed that the
contribution of EC sodium to the increase in sodium image intensity is
negligible. As to potential explanation a, a reduction in IC
T1 (or increase in EC
T1) could also contribute to increased
image intensity, and one could postulate that large changes in[
Na]i by themselves could alter IC
T1. However, during the intervention
of hypoxia or ouabain poisoning, which can dramatically increase[
Na]i, IC T1
values were reported stable (43)
. Likewise, IC
T2 values are stable during pump
blockade in the heart (21)
. Thus, although such alternate
hypothesis motivate additional studies, it seems unlikely that such
results could alter the diagnostic potential of this measurement.

Absolute quantitation, although a long-term goal of this approach, is
confounded by various issues, not the least of which is tissue,
cellular, and subcellular compartmentalization. For example, diffusion
on a molecular level could result in an exchange of sodium between
molecular domains with different T1
values during the recovery from the inversion pulse. This would be the
case if the diffusion relaxation time constant were comparable with
inversion time. As has been shown in submicron vacuoles
(44)
, this exchange process leads to an averaging of the
T1 values and interferes with the
ability to distinguish sodium inside and outside the vacuole membrane.
However, the size of tumor cells and the limited unidirectional
exchange through the polarized cell membranes make substantial exchange
between IC and EC compartments during an inversion pulse unlikely
(45, 46)
. Furthermore, measured values of
T1 in mammalian IC and EC spaces show
primarily single exponential relaxation kinetics. The EC
T1 value is longer, consistent with
our hypothesis. Sodium localized to regions of the interstitial spaces,
where there are more numerous binding sites, do have short
T1s (47)
, but (as
above) there is no evidence that they or T1s of
the IC contribute a changing component to the IR image (43, 48)
.

The correlation between apoptosis and IR image intensity is apparent in
comparing the histology and images. Tumors with significant fluid
spaces or large centers with cellular debris had dark centers on the IR
weighted images. In this study, the only tumor with no IR signal had a
narrow rim of surviving cells, a large nonviable center with
significant fluid, but no significant interface with back-to-back
apoptosis. (No chemotherapy was administered for this tumor.) Tumors
with bright annuli had sizeable rims of back-to-back apoptosis. Tumors
with bright centers had back-to-back apoptosis extending into the
central region with minimal nonviable tissue. Although we could not do
a time study of the histological parameters, the results are
consistent with the notion that the brightness comes from dense regions
of apoptosis and/or early apoptotic processes in the surviving rim. On
the basis of the spatial resolution of the image (∼1 mm within each
slice), the enhanced intensity could be from a spatial broadening of
the apoptotic rim, an increased density of apoptotic cells (including
early commitment to apoptosis), or both.

Apoptosis is a dynamic process, which ends with complete fragmentation
and loss of cell nuclei and most of the other cellular macromolecules.
Hence, the absence of a weighted IC sodium MRI signal in late apoptosis
can be explained by the loss of polyanions, because the IC space
equilibrates its chemical composition with the EC fluid. This
prediction is consistent with our finding (Fig. 4)<$REFLINK>
that the average IR
intensity increase is smaller at 48 than at 24 h. A more detailed
quantitative analysis is required to correlate specific cell
populations with image responses and specific ionic responses with
molecular events induced by the chemotherapy.

Because proton MRI, which uses the hydrogen nucleus, is best suited for
structural studies and angiography, we have chosen to develop clinical
diagnostic approaches using sodium MRI because of the major involvement
of sodium in important dynamic processes in the cell, one of which,
illustrated here, is the interaction of chemotherapy, cellular
apoptosis, and ions. The ease with which sodium images can be
coregistered with proton high resolution images is a further advantage
of this technique, as is the fact that it does not require costly or
potentially toxic reagents, as do traditional SQ sodium MRI or the new
techniques used in gene marker or receptor imaging.

Acknowledgments

The helpful discussions of Prof. I. Bernard Weinstein and Robert
DeLaPaz are greatly appreciated. We acknowledge the roles of K. J.
Jung, Supryo Das, Kenny Hess, and Daniel Mollura for participating in
these experiments. Richard Sano assisted in image analysis and
presentation. R. P. K. acknowledges the contribution of Walter
Suskind.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

↵1 This work was supported in part by a grant from
Rhone-Poulenc Pharmaceuticals, Inc. and the Rose M. Badgeley Residuary
Charitable Trust.